key: cord-323654-9nnjex9y
authors: Ramachandran, Ashwin; Huyke, Diego A.; Sharma, Eesha; Sahoo, Malaya K.; Banaei, Niaz; Pinsky, Benjamin A.; Santiago, Juan G.
title: Electric-field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2
date: 2020-05-22
journal: bioRxiv
DOI: 10.1101/2020.05.21.109637
sha: 
doc_id: 323654
cord_uid: 9nnjex9y

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In a basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex is activated when it highly specifically binds to target DNA, and the activated complex non-specifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We recently discovered that electric field gradients can be used to control and accelerate this CRISPR assay by co-focusing Cas12-gRNA, reporters, and target. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab sample. We here combine this ITP purification with loop-mediated isothermal amplification, and the ITP-enhanced CRISPR assay to achieve detection of SARS-CoV-2 RNA (from raw sample to result) in 30 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables a new modality for a suite of microfluidic CRISPR-based diagnostic assays. Significance statement Rapid, early-stage screening is especially crucial during pandemics for early identification of infected patients and control of disease spread. CRISPR biology offers new methods for rapid and accurate pathogen detection. Despite their versatility and specificity, existing CRISPR-diagnostic methods suffer from the requirements of up-front nucleic acid extraction, large reagent volumes, and several manual steps—factors which prolong the process and impede use in low resource settings. We here combine on-chip electric-field control in combination with CRIPSR biology to directly address these limitations of current CRISPR-diagnostic methods. We apply our method to the rapid detection of SARS-CoV-2 RNA in clinical samples. Our method takes 30 min from raw sample to result, a significant improvement over existing diagnostic methods for COVID-19.

We next developed a novel protocol for the detection of RT-LAMP-amplified cDNA of 151 SARS-CoV-2 viral RNA using ITP-mediated CRISPR-Cas12 DNA detection. Upon The LOD of the ITP-CRISPR method was found to be 10 copies per microliter reaction, 165 which is the same as the very recent CRISPR-based assay (3). Further, in the case of 166 positive detection, a fluorescence signal above the threshold value was observed in < 3 167 min (Fig. S7) . These results are in contrast to the 1 copy per microliter reaction LOD for 168 the 2-hour qPCR method (Fig. 2e) (1). Lastly, we verified that microfluidic ITP-CRISPR 169 detection and the typical CRISPR-based (3) approaches gave the same positive/negative 170 result when tested with the same LAMP pre-amplified DNA (Fig. S8) . 171 172 ITP enables rapid extraction of total nucleic acids from raw nasopharyngeal swab 173 samples 174

We also demonstrated on-chip ITP extraction of total nucleic acids from raw clinical 175 positive and negative nasopharyngeal swab samples (Figs. 1b, 2a and 2d ). To validate 176 our extraction method, we performed qPCR for the E gene and RNase P control (Fig. 2e) . 177

We observed that ITP-extracted nucleic acids showed E gene amplification on positive qPCR-based detection approaches is provided in Table 1 . 192 In summary, we developed an electrokinetic microfluidic method broadly applicable to 194 CRISPR-based diagnostics. Our method involves ITP-based nucleic acid extraction from 195 raw sample, isothermal reverse transcription and amplification, and then a novel CRISPR 196 assay enhanced by ITP with a total assay time of 30 min (from raw sample to result). We Table S1 . LAMP primers (Elim Biosciences) were reconstituted in nuclease free water 276 and gRNAs (IDT) were reconstituted in RNA reconstitution buffer. 277

For ITP co-focusing visualization experiments, the Mtb target DNA sequence was used 278 (Table S1 ). 1 μM stock solution of Mtb dsDNA was prepared by pre-hybridizing 279 complementary ssDNA templates (Elim Biosciences) in a buffer containing 50 mM Tris-280

HCl, 5 mM MgCl2, and 1 mM EDTA at 37 o C. We designed a Cy5-labeled gRNA (IDT, 281

Table S1) to target the Mtb dsDNA sequence. main channel length between the positive/negative electrodes is 72 mm (Fig. S10) . To 297 avoid cross-contamination, ensure run-to-run repeatability, and provide uniform surface 298

properties, the channels were rinsed in the following order before each ITP experiment: 299 10% bleach for 2 min, DI water for 2 min, 1% Triton-X for 2 min, DI water for 2 min, 1 M 300 NaOH for 2 min, and DI water for 2 min. Between each rinse step, the channel was 301 completely dried using vacuum. The buffer loading procedure and buffer placement in the 302 channel sections are detailed in Fig. S10 . 1x composition of lysis buffer included 1.5 % Triton X, 1 mg/mL of Proteinase K, 310 0.1 mg/mL of carrier RNA (Thermo Fisher). Following incubation, 1 μL of 300 mM HEPES 311 buffer was added, and 10 μL of this mixture was dispensed in the trailing electrolyte (TE) 312 reservoir on-chip (Fig. S10) . The leading electrolyte (LE) buffer in the main channel 313 consisted of 100 mM Tris-HCl (pH 7.5), 1 U/μL RNasin Plus, 0.2 % Triton X, 1% of 1.3 314

MDa Polyvinylpyrrolidone (PVP) and 1x SYBR Green I. SYBR Green I was used to 315 visualize the ITP peak which contained nucleic acids (Fig. 2d) 

A 10x Cas12-gRNA complex mixture was prepared by pre-incubating 1 μM of LbCas12a 336 (NEB) with 1.25 μM gRNA in 1x NEBuffer 2.1 at 37°C for 30 min. LbCas12-gRNA 337 complexes were prepared independently for N, E, and RNase P genes. 338

For ITP co-focusing visualization experiment in Fig. 1c, a 10x Cas12-gRNA complex was 339 prepared using 1 μM of LbCas12a (NEB) and 0.5 μM of Cy5-labeled gRNA. Here, a molar 340 excess of LbCas12a was used to minimize free, unbound gRNA. LbCas12-gRNA complex was combined with 2 μL of pre-prepared Mtb dsDNA template 350 and 16 μL LE buffer. The on-chip buffer loading procedure is described in Fig. S10 . 351

The ITP-CRISPR detection experiments were performed at constant current of 4 μA 352 supplied by a Keithley 2410 sourcemeter (Fig. S11) . Fluorescence images of the moving 353 ITP peak were acquired in 30 s intervals using a CMOS camera (Hamamatsu ORCA-354

Flash4.0) mounted on an inverted epifluorescence microscope (Nikon Eclipse TE200). 355

For widefield images of ITP peak in Fig. 1b, 

The RT-qPCR assay was performed using the ABI 7500 Fast DX (Applied Biosystems) 378 instrument. We performed assays for the E and RNase P genes separately in 20 µL 379 reaction volumes using the Luna Universal Probe One-Step RT-qPCR Kit (New England 380 deviation. Covid19-P4 was below the LOD of our assay as confirmed by qPCR (Fig. 2e) . is loaded in reservoir 6, 5 μL of LE is loaded in reservoir 8, and 5 μL TE combined with 500 ssDNA reporters is loaded in reservoir 7. Vacuum is applied at reservoir 5 briefly till the 501 channels are filled as depicted in the schematic. Then, reservoirs 5, 6, and 8 are emptied 502 and loaded with 10 μL of LE, and reservoir 7 is emptied and loaded with 10 μL TE. A 503 constant current of 4 μA is applied for 5 min and fluorescence intensity of the ITP peak is 504 recorded using a CMOS camera every 30 s. 505 (14) for an integration of at least one ITP assay into a portable device. We propose here 520 the concept that such a system can integrate ITP-based nucleic acid extraction, 521 multiplexed isothermal amplification of target cDNA of N and E genes of SARS-CoV-2 522 and RNase P control, followed by ITP-CRISPR-based cDNA detection in three separate 523 channels using photodiodes. 524 525  Table S1 . List of gRNAs, LAMP primers, RT-qPCR primers, template and reporter 526 sequences. Mtb sequences were used for ITP co-focusing experiments of Figure 1c 

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